The Notch signaling pathway is remarkably important for the development and maintenance of most complex tissues in multicellular organisms and defective Notch signaling has been implicated in severe developmental defects and cancer. Understanding the basic mechanisms and functions of Notch is imperative to developing therapies for Notch-related diseases. Notch is a single-pass transmembrane receptor that, upon activation by a ligand, is cleaved in the transmembrane domain to release the intracellular domain into the cytoplasm. The free intracellular domain translocates to the nucleus where it forms a ternary protein complex to promote target gene transcription and influence cell fate and function. The Notch intracellular domain is difficult to visualize directly because the free intracellular domain is rapidly degraded, but manipulations affecting the stability or level of the intracellular domain cause cell fate transformations, complicating studies of signaling onset, duration, and dynamics. Thus, Notch activity has been assessed using target gene transcriptional reporters, which are limited by time lags due to transcription, translation, and fluorescent protein maturation, as well as perdurance of reporter proteins after signaling has ceased. Preliminary data shows that a Sensor Able to detect Lateral Signaling Activity, or SALSA, is a promising novel, rapid-response, in vivo biosensor of Notch activity. SALSA is a bipartite system consisting of a ?switch? in which the Notch locus is endogenously tagged with a protease, and a cleavable, dual-fluorescent protein reporter. When Notch is activated, the protease- tagged intracellular domain translocates to the nucleus where the protease cleaves the reporter and that allows the individual fluorescent proteins to be localized to different cellular compartments. This project will 1) establish a protocol for quantifying the level of Notch activity using SALSA. This protocol will be established in model organism C. elegans because of the speed of generating transgenic lines, the ability to image the entire organism in vivo throughout its lifecycle, and the well conserved and characterized Notch signaling events. This project will then 2) use SALSA to determine how the timing and levels of Notch activity influence cell fate decisions in two paradigms for Notch signaling in C. elegans: the Vulval Precursor Cells and the Anchor Cell/Ventral Uterine precursor cell decision in the somatic gonad. Results from this project will establish a useful tool for studying many aspects of Notch signaling in a variety of contexts, and provide insight into a universal relationship between the timing and levels of Notch activity with cell fate decisions.